Enzymes are powerful tools for synthetic chemistry not only because they can catalyze reactions with exceptional levels of reactivity, but also because they can be tuned using directed evolution to achieve high selectivity in situations that would be intractable for small-molecule catalysts. Enzyme engineering has also enabled the heme-dependent monooxygenase cytochrome P450BM3, to catalyze carbene transfer reactions between diazoesters and mild nucleophiles, a reaction type that is completely absent in nature. The focus of this proposal is to expand the synthetic utility of this methodology by using directed evolution of P450BM3 to develop carbene transfer catalysts that can synthesize biologically important molecular scaffolds that are difficult to access with existing methods. The specific aims are: (1) to evolve P450BM3 variants to synthesize enantioenriched di- and triarylmethanes via asymmetric carbene insertion into N-H and O-H bonds; and (2) to extend the substrate scope to include electron-rich arenes and apply this methodology to the asymmetric synthesis of 3,3-disubstituted indole and ?,?-disubstituted glycine derivatives. The di- and triarylmethane motif is prevalent in numerous synthetic drug candidates, but is difficult t synthesize asymmetrically, partly because the area surrounding the chiral center has local symmetry that makes enantioselection difficult for small-molecule catalysts. The use of enzymes is advantageous, however, because an enzyme's active site creates a chiral environment that interacts with the entire structure of the substrate. In the second aim, the application of carbene transfer to electron-rich arenes constitutes a novel approach to enzyme-catalyzed C-C bond formation, which is usually accomplished with aldolases. The indole and glycine derivatives that we propose to make with this method are found in numerous bioactive natural and synthetic compounds, and thus there is continued demand for new methods to synthesize these structures asymmetrically. We will characterize the enzymes that result from this project with a variety of techniques, such as X-ray crystallography, in order to elucidate the mechanisms of selectivity and assist in the future development of enzyme catalysts for other reaction types.